Transdermal delivery system of nanostructured lipid carriers loaded with Celastrol and Indomethacin: optimization, characterization and efficacy evaluation for rheumatoid arthritis

Figures & data

Figure 1. The structures of NLCs and preparation process of Cel-Indo-NLCs-gel.

Table 1. Factors and levels for the experimental design.

		Codes/levels
Factors		–1	0	1
Independent variables	A, concentration of total lipids (% w/v)	3	5	7
	B, concentration of surfactant (% w/v)	1	2	3
	C, amount of added total drugs (mg/g)	1	2	3
Dependent variables	Y1, particle size (nm)
	Y2, the average entrapment efficiency (%EE)
	Y3, the total drug loading (%DL)

The ratio of added Celastrol and Indomethacin was 1:1 and the ratio of solid lipid (Precirol ATO 5) and liquid lipid (Labrasol ALF) was 7:3.

Table 2. Randomized design and results for Box–Behnken design of total 17 experiments.

Runs	Independent variables			Dependent variables
	A	B	C	Y1	Cel		Indo		Y2	Y3
				size	EE	DL	EE	DL	Mean EE	Total DL
				nm	%	%	%	%	%	%
1	1	1	0	17.03	93.03	1.28	95.04	1.37	94.06	2.65
2	0	0	0	61.05	95.00	1.86	98.15	1.84	96.58	3.70
3	0	0	0	137.3	98.80	1.98	95.37	1.90	97.09	3.88
4	0	–1	–1	195.2	94.00	0.95	99.43	1.00	96.72	1.95
5	0	0	0	78.79	96.69	1.94	93.50	1.88	95.09	3.82
6	1	–1	0	216.8	83.78	1.22	84.27	1.22	84.03	2.44
7	–1	1	0	22.39	80.26	3.45	80.15	3.26	80.20	6.71
8	0	1	–1	14.55	98.71	1.00	92.36	0.92	95.53	1.92
9	0	1	1	81.69	96.33	2.90	92.31	2.80	94.32	5.70
10	0	–1	1	109.9	79.80	2.38	80.93	2.43	80.37	4.81
11	0	0	0	79.4	100.85	2.04	98.82	1.99	99.83	4.03
12	–1	0	1	48.01	80.77	4.09	89.53	4.52	85.15	8.61
13	–1	–1	0	51.78	74.23	2.83	90.20	3.00	82.21	5.83
14	1	0	–1	19.88	91.32	0.69	88.03	0.65	89.67	1.34
15	1	0	1	140.8	92.13	1.97	88.82	1.91	90.47	3.88
16	–1	0	–1	109	86.14	1.64	84.16	1.61	85.15	3.25
17	0	0	0	89.66	97.50	1.59	98.80	1.55	98.15	3.14

Figure 2. Equilibrium solubility of Cel and Indo in liquid (A) and solid (B) lipids.

Table 3. Summary of the results of regression analysis for responses, and analysis of variance for particle size, mean EE and total DL.

	Model	Sum of squares	df	F-value	p Value	Mean	SD	C.V.%	R^2
Size	2FI	48875.00	6	9.55	.0012	86.66	29.21	33.70	0.8514
Mean EE	Quadratic	615.34	9	8.20	.0056	90.86	2.97	3.27	0.9134
Total DL	Quadratic	55.99	9	57.26	<.0001	3.98	0.33	8.28	0.9866

Table 4. Predicted and experimental values obtained based on optimal formula generated by BBD (n = 3).

	Factors			Responses
	A	B	C	Size	Mean EE	Total DL
Predicted values	5%	3%	2 mg/g	29.91 nm	95.81%	3.87%
Experimental values	5%	3%	2 mg/g	26.92 ± 0.62 nm	96.56 ± 1.36%	3.65 ± 0.05%
RSD (%)				2.31	1.41	1.32

Figure 3. Size (A) and TEM (B) images of optimized Cel-Indo-NLCs.

Figure 4. Fourier transform infrared spectra of Indo, Cel, Mixture (mixture of drugs and lipids), Blank-NLCs and Cel-Indo-NLCs.

Figure 5. Powder X-ray diffraction patterns of Indo, Cel, Mixture (mixture of drugs and lipids), Blank-NLCs and Cel-Indo-NLCs.

Figure 6. CLSM images of vertical section of the skin treated with Cel-Indo-NLCs at different time. The slides were stained with DAPI. White arrows indicate the hair follicles.

Figure 7. CLSM images of horizontal section of skins treated with Cel-Indo-NLCs at 24 h. The slides were stained with DAPI.

Figure 8. The transdermal delivery mechanism and depth of NLCs into skin.

Figure 9. Rheological profiles of Cel-Indo-NLCs-gel.

Figure 10. In vitro permeation profiles of Cel and Indo from Cel-Indo-NLCs and Cel-Indo-NLCs-gel through excised skins of rats.

Figure 11. Effects of different administrations on paw volumes (n = 6).

Figure 12. Effects of different treatments on serum levels of inflammatory and pain factors in rats. **p < .01, *p < .05 compared with the model group (n = 6). A, B, C, D, E and F respectively represent Normal (normal rats), Model (RA rats without treatment), Safety control (normal rats with Cel-Indo-NLCs-gel), Cel-NLCs-gel (RA rats with Cel-NLCs-gel), Indo-NLCs-gel (RA rats with Indo-NLCs-gel) and Cel-Indo-NLCs-gel (RA rats with Cel-Indo-NLCs-gel) group.

Figure 13. Effects of different administrations on paw appearances, histopathological images of ankle joints and skins. The degree of severity of inflammation is presented as 0 = normal; + =mild; ++=moderate; +++ =severe. The images presented are dedicated for (A) Normal (normal rats), (B) Model (RA rats without treatment), (C) Safety control (normal rats with Cel-Indo-NLCs-gel), (D) Cel-NLCs-gel (RA rats with Cel-NLCs-gel), (E) Indo-NLCs-gel (RA rats with Indo-NLCs-gel) and (F) Cel-Indo-NLCs-gel (RA rats with Cel-Indo-NLCs-gel) group.

Figure 14. Histopathological images of kidneys and testis of different groups. The images presented are dedicated for (A) Normal (normal rats), (B) Model (RA rats without treatment), (C) Safety control (normal rats with Cel-Indo-NLCs-gel), (D) Cel-NLCs-gel (RA rats with Cel-NLCs-gel), (E) Indo-NLCs-gel (RA rats with Indo-NLCs-gel) and (F) Cel-Indo-NLCs-gel (RA rats with Cel-Indo-NLCs-gel) group.